JP3277523B2 - Factor Xa linker DNA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene corresponding to a recognition amino acid sequence of blood coagulation factor Xa having a restriction protease activity, a fusion gene containing the same, an expression vector,
The present invention relates to Escherichia coli carrying the expression vector and methods for producing them .

[0002]

2. Description of the Related Art In a genetic engineering technique for introducing a foreign gene into a host cell such as Escherichia coli to express a protein,
It is effective to insert a target gene into the middle of a structural gene in an existing high-efficiency expression vector to obtain a large amount of a fusion protein (for example, Molecular Cloning such as Maniatis). Laboratory Manual (A Laboratory Manual), 2nd
Edition, pp. 17.3-17.9, Cold Spring Harbor
Laboratory Press (Cold Spring Harbor Laboratory P)
ress), 1989). At that time, various chemical or enzymatic techniques have been developed to specifically cleave only the fusion site of the fusion protein in order to isolate the target mature protein (for example, D.V.Jodel (DV) G
oeddel), Method in Enzymology (Method
s in Enzymology), 185 , 129-143 (1990)). inside that,
Specific recognition of the amino acid sequence Ile-Glu-Gly-Arg
Attention has been paid to a technique using a blood coagulation factor Xa having a protease activity for cleaving the C-terminal side. A typical example is the restriction enzyme Bam in the structural gene of an existing expression vector.
The purpose of inserting a DNA linker containing a nucleotide sequence corresponding to the factor Xa recognition amino acid sequence (hereinafter abbreviated as Xa linker DNA) into the HI cleavage site and blunting the 5 'end to the restriction enzyme Stu I cleavage site inside the linker And treating the expressed fusion protein with factor Xa to obtain the desired mature protein (Koshinagai et al., JP-A-61-135591). In addition, examples of fusion of Xa linker DNA upstream of the human B cell differentiation factor gene (Yoshihiro Asakoshi et al.,
JP-A 2-2353), an example in which Xa linker DNA is fused upstream of the human growth hormone gene (Geat Grandy et al., JP
-2386), and an example in which a gene encoding several residues of glycine is linked before and after Xa linker DNA (Andreas Brucktown, JP-A-2-5881).

[0003]

There are two problems in fusing Xa linker DNA with a foreign gene of interest. One is to fuse the codons so that the reading frames are aligned, and the other is to generate a mature foreign protein when the fusion protein is cleaved with factor Xa. Japanese Patent Application Laid-Open No. 61-135591 deals with a general method of fusion with a foreign gene in the above disclosed examples. In this case, the fusion site is the recognition sequence AGGCCT of the restriction enzyme Stu I inside the Xa linker DNA, and the Arg codon AGG forms the 3 ′ blunt end of the fusion upstream portion by cleavage with Stu I. Therefore, in order to achieve the above two tasks by this method, it is necessary to prepare a foreign gene so that the 5 ′ terminal base is blunted to become the first letter of the codon.

[0004] However, it is generally difficult to smooth the 5 'end of the foreign gene to be fused so as to correspond to the first letter of the codon, and the probability is one-third. In other cases, it is impossible to use the vector of the above-mentioned typical example as it is.

[0005] One object of the present invention is to insert a DNA encoding a factor Xa recognition sequence upstream of an inserted gene which has been subjected to blunting to generate a 5 'end with one nucleotide added to the codon reading frame. An object of the present invention is to provide a linker DNA in which codon reading frames are aligned and ligated.

Another object of the present invention, for any structural gene having specific restriction enzyme sites, the production of the fusion gene and its <br/> linked a foreign gene of interest via the Xa linker DNA Is to provide a way.

[0007]

According to the present invention, the Factor Xa linker DNA contains the recognition sequence TCGCGA of the restriction enzyme Nru I, and corresponds to the fourth amino acid sequence recognized by Factor Xa by cleavage with Nru I. A blunted fusion upstream containing up to the second character (CG) of the Arg codon is obtained. Therefore, a fusion gene with a uniform reading frame is provided by ligating the 5 'end of the foreign gene to be fused to the upstream portion with an additional nucleotide 5' more than the reading frame of the codon added thereto. . Since the first and second characters of the Arg codon in this case are CG, this codon encodes 3
The nucleotide in the letter may be any of A, C, G, and T, and the nucleotide added to the 5 'end of the foreign gene to be fused may be arbitrary.

In general, the probability that the 5 'end of a foreign gene is blunted to a state in which one extra nucleotide is added to the codon reading frame by one nucleotide is one-third. However, due to recent advances in genetic engineering, the number of cases where the whole or part of a target gene is chemically synthesized has increased, and in that case, a recognition site for a restriction enzyme is often provided at the 5 'end of the gene. Therefore, a method for modifying a foreign gene having a restriction enzyme recognition site at the 5 ′ end to a form that can be used in the present invention will be described with reference to examples. First, immediately before the first codon of a foreign gene, there is a restriction enzyme that recognizes 6 bases represented by BamHI, EcoRI, HindIII, PstI, SacI, etc. and forms a 4 nucleotide protruding end after cleavage. (Fig. 3a), after cleavage with the restriction enzyme, the protruding ends are removed with Mung Bean nuclease or S1 nuclease, so that one nucleotide is added immediately before the first codon of the foreign gene, and this remains Arg. The third character can be composed.

Next, when the restriction enzyme Nde I recognition sequence CATATG or NcoI recognition sequence CCATGG containing the initiation codon ATG is present at the 5 'end of the foreign gene (FIG. 3b), after digestion with the restriction enzyme, Fill the protruding ends with T4 DNA polymerase.
By increasing the number, one nucleotide is added immediately before the start codon Met , and a gene having a blunt 5 ′ end can be prepared. In this case, exogenous protein having an initiation codon Met added to the N-terminus can be obtained by factor Xa cleavage after expression of the fusion protein. The scope of the present invention is not limited to the examples given here, but can be applied to genes having many restriction enzymes at the 5 'end.

[0010] Further, in the present invention, the factor is added to both of the double-stranded DNA.
To provide information on the Xa recognition sequence, as shown in FIG.
Xa linker DNA containing a 22-base pair inverted repeat sequence centered on the uI recognition site is provided. Further according to the present invention, by providing an appropriate restriction enzyme recognition sequence or restriction enzyme cleavage sequence on both sides of the Factor Xa linker DNA,
The factor Xa linker DNA can be inserted into any structural gene having a specific restriction enzyme recognition site. For example, in the Xa linker DNA shown in FIG. 1, a recognition sequence of Eco RI and Bam HI is added to the outside of the Xa recognition sequence , and the linker is inserted into an arbitrary structural gene containing a recognition site of Eco RI and Bam HI. Can be inserted. in this case,
Due to the inverted repeatability of the linker, Eco RI in the structural gene
And the order of the Bam HI recognition sites does not matter. Further, in the Xa linker AACGAATTCA TCGAGGGTCG CGACCCTCGA TGAATTCGTT structure having a recognition sequence of Eco RI outside both sides of Xa recognition sequence, the linker optional structural gene but recognition site comprises does not include the recognition sequence of Bam HI of Eco RI Can be inserted.

[0011]

According to the present invention, the factor Xa linker DNA is ligated into the structural gene in the expression vector using a restriction enzyme or the like so that the codon reading frames are aligned, and then the restriction enzyme Nru in the Xa linker DNA is used. By ligating the foreign gene of interest to the I recognition site so that the reading frames of codons are aligned, a vector containing a fusion gene ligated with the foreign gene of interest via Xa linker DNA can be obtained. Further, by culturing Escherichia coli transformed with the expression vector, a fusion protein corresponding to the fusion gene is produced. Further, the fusion protein is recovered, and factor Xa having a restricted protease activity is obtained.
, A mature form of the foreign gene expression protein can be obtained.

[0012]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it should not be construed that the present invention is limited thereto. The restriction enzymes used in this example were manufactured by Takara Shuzo Co., Ltd.
Manuals (MolecularCloning: A Laboratory Man)
ual, 2nd edition, Cold Spring Harbor Laboratory Press, 1
989).

Example 1 Coagulation Factor Xa Linker DNA
Synthesis and Cloning The two 40-base oligonucleotides shown in FIG.
A synthesizer (Applied Biosystems,
It was synthesized by the phosphoramidite method using Model 380B). After adding ammonia water and leaving at 55 ° C. for 12 hours to remove the protecting group, OPC (oligonucleotide purifica
Purification was carried out using a column with a cartridge. Further, after the solvent was distilled off under reduced pressure under a centrifugal pressure, the two solidified oligonucleotides were respectively dissolved in 100 μl of TE buffer, and the absorbance at 260 nm was measured to carry out a concentration test. Subsequently, the thus purified and quantified oligonucleotide was fractionated by 0.1 nmol each, and 50 mM Tris-HCl (pH 8.0), 10 mM MgC
The phosphorylation reaction of the 5 'end was performed at 37 ° C for 1 hour in l2, 10 mM _2- mercaptoethanol, 1 mM ATP, and 20 U of T4 polynucleotide kinase (manufactured by Toyobo Co., Ltd.). Thereafter, the two were mixed and subjected to phenol treatment and ethanol precipitation, and the collected DNA was subjected to ligation buffer (100 mM Tris-HCl (pH 7.
6), dissolved in 10 μl of 6.5 mM MgCl ₂ ), left at 70 ° C. for 15 minutes, and then slowly annealed to room temperature. By the above operation, about 10 ng / μl of factor Xa linker DNA was obtained.

Subsequently, according to FIG.
The method for preparing a plasmid containing First, the plasmid vector pTRL disclosed in Japanese Patent Application No. 1-260261.
ORF1 (deposited as No. 11043 (FERM P-11043)) was cleaved with the restriction enzyme HpaI having a recognition site in trp P / O and the gene ORF1, as shown in FIG. . Then, in order to prevent self-ligation of the vector, 4.58 kilobase pairs using alkaline phosphatase of E. coli (manufactured by Toyobo Co., Ltd.)
After performing 5'-terminal dephosphorylation of the Hpa I fragment of (kb), 100 ng equivalents were collected, and a ligation reaction with the above factor Xa linker DNA (30 ng) was performed at 16 ° C. using a ligation kit (manufactured by Takara Shuzo Co., Ltd.). We went for 8 hours. Escherichia coli competent cell DH5 (manufactured by Toyobo Co., Ltd.) was transformed with 10 μl of the reaction solution to obtain recombinant Escherichia coli DH5 [pTRXa-1] and DH5 [pTRXa-2]. Here, pTRXa-2 refers to pTRXa-1 shown in FIG.
Is a plasmid in which the orientation of the [Hpa I-Hpa I] insert is reversed. This Escherichia coli E. coli DH5 [pTRXa-1] was deposited at the Institute of Microbial Industry and Technology (hereinafter, abbreviated as “Microtechnical Laboratories”) under the name of Microorganism Bacteria No. 12501 (FERM P-12501). I have.

The linker DNA shown in FIG.
The recombinant vectors pTRXa-1 and pTRXa-2 produced here were designed to be the cleavage ends of
It has the characteristic that the Xa linker DNA can be recloned into any other vector that can be recut with I and can form blunt ends.

Subsequently, according to FIG. 1, the Xa linker DN
A method for inserting A into an existing expression vector pGEMEX-1 (Promega) will be described. pGEMEX-1 encodes a T7 phage-derived promoter and T7 major head protein.
It contains 10 genes and has a multiple cloning site (MCS) for restriction enzymes in the gene. This vector is used for Eco RI in MCS.
And Bam HI, and the ends were dephosphorylated using alkaline phosphatase. On the other hand, the vector pTRXa-1
The Xa linker DNA fragment was recovered by cleavage with coRI and BamHI, and ligated with the above vector using a ligation kit. Then, using this reaction solution, E. coli competent cell DH5 was transformed to obtain recombinant E. coli DH5 [pT7Xa].

Example 2 T7 via Factor Xa Linker DNA
Fusion of phage 10 gene and octopus rhodopsin fragment gene
Construction of a Syngene A target foreign gene was inserted into the NruI recognition site of the Xa linker in the expression vector pT7Xa prepared in Example 1 to allow the T7 tenth gene and the foreign gene to be mediated via the factor Xa linker DNA. And a fusion gene can be constructed. Here, as an example, Japanese Patent Application No. 1-26 according to FIG.
[0261] A method of fusing a fourth fragment gene, which is a partial gene of the octopus rhodopsin gene disclosed in the above, to the expression vector will be described.

First, the vector pTRLORF4 containing the octopus rhodopsin fourth fragment gene was digested with EcoRI, followed by Mung Bea
n Using a nuclease (Takara Shuzo Co., Ltd.)
T) was removed to blunt the 5 'end. After removing Mung Bean nuclease by phenol treatment, Bam HI
And the ORF4 portion of the fourth fragment gene was recovered.
On the other hand, the expression vector pT7Xa was cut with Nru I and Bam HI, and the 5 ′ end was dephosphorylated using alkaline phosphatase. Both were ligated using a ligation kit, and Escherichia coli DH5 was transformed with the reaction solution to obtain recombinant Escherichia coli DH5 [pT7XaoRh4]. This E. coli E.coliD
H5 [pT7XaoRh4] was sent to NIKEN by NIKEN Bacteria No. 12500 (FERM
P-12500).

As shown in FIG. 2, the 3 'end of the Xa linker in the vector pT7Xa cut with Nru I corresponds to the second letter of the Arg codon, while the 5' end of the ORF4 to be fused is in the codon reading frame. Since the blunting was performed in a form in which nucleotides were added, a fusion gene having a uniform reading frame was constructed by ligating these.

Example 3 Expression of Fusion Gene and Factor Xa
First production of mature protein that was the host E. coli JM109 (DE3) for expression in an expression vector pT7XaoRh4 prepared in Example 2 was transformed. A single colonized recombinant Escherichia coli JM109 (DE3) [pT7XaoRh4] was cultured overnight at 37 ° C. in 10 ml of NZCYM medium containing 50 μg / ml of ampicillin, and 0.5 ml of the resulting culture solution was treated with 50 μg of ampicillin NZC / ml NZC.
Cultured at 37 ° C in 100 ml of YM medium, absorbance at 600 nm is 0.
When it reaches 9, Isopropyl-β-D-thio-Galactopy
ranoside (IPTG, manufactured by Wako Pure Chemical Industries, Ltd.)) was added to a final concentration of 1 mM, and the cells were cultured overnight. After completion of the culture, the culture solution is centrifuged to collect the cells, and a 10 mM sodium phosphate buffer solution (pH: 150 mM, containing sodium chloride and 0.02% (W / V)
7.0) The cells were resuspended in 10 ml and the cells were disrupted using a French press. The crushed liquid is centrifuged at 3,000 G for 15 minutes to separate the supernatant and precipitate (Fraction 1) .The supernatant is further centrifuged at 180,000 G for 1 hour to separate the supernatant (Fraction 2) and precipitate (Fraction 3). Was. Fractions 1, 2, and 3 correspond to insoluble granule proteins, cytoplasmic proteins, and membrane proteins, respectively. Sample each of these fractions,
When subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at a gel concentration of 16.5%, fraction 1 had a molecular weight of about 4
Ten thousand bands were detected. Therefore, via the Xa recognition sequence,
It was found that the fusion protein of the T7 major head protein and the octopus rhodopsin fourth fragment protein was accumulated as insoluble granules in Escherichia coli.

Fraction 1 was rinsed five times with 0.5% Triton X-100 and 1 mM EDTA solution, and dissolved in 5 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 8 M urea, 1 mM EDTA, and 1 mM dithiothreitol. Subsequently, the resultant was dialyzed against 100 mM sodium chloride and 50 mM Tris-HCl buffer (pH 8.0) for 18 hours. When the inner solution of the dialysis was centrifuged, no precipitate was found. The concentration of the crude solution of the fusion protein was determined by the Bradford assay (Analytical Biochem. 72 , 248 (197
It was estimated to be about 10 mg / ml according to 6)).

1 μl of the crude purified protein was added to a reaction buffer (1
00 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM CaCl ₂ ) 8.5 μl
And restriction protease factor Xa (1 μg / μl, Takara Shuzo
(Manufactured by Co., Ltd.) was added thereto and reacted at 25 ° C. for 24 hours. When the reaction solution was subjected to the above-mentioned SDS-PAGE at a gel concentration of 16.5%, most of the cleavage reaction had progressed, and a band corresponding to the mature thacorhodopsin fourth fragment was detected near 7 kDa. This band is blotted on a clear blot-P membrane (manufactured by ATTO Co., Ltd.), and a protein sequencer (Applied Biosystems, Model 477A) is used.
Was used to confirm that the amino acid sequence of the N-terminal 10 residues matches the mature octopus rhodopsin fourth fragment protein.

[0023]

According to the present invention, it has become possible to fuse a foreign gene into a structural gene in an expression vector containing a promoter / structural gene having high expression efficiency via the Xa linker DNA. Since a foreign gene, particularly a gene of a higher animal or plant, cannot often be expected to have high expression efficiency with a vector directly ligated downstream of the promoter / Shine-Dalgarno (SD) sequence, a fusion gene is prepared according to the present invention, and a large amount of the gene is produced in E. coli. This method, which is expressed as a fusion protein and is cleaved with factor Xa to obtain a mature protein, is an effective means that greatly contributes to expanding the scope of genetic engineering.

[0024]

[Sequence list]

SEQ ID NO: 1 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: cleavage-site Location : 4..9 Method to determine the feature: S Symbol representing the feature: cleavage-site Location: 18..23 Method to determine the feature: S Symbol to represent the feature: cleavage-site Location: 32..37 Feature Determination method: S sequence AACGAATTCA TCGAGGGTCG CGACCCTCGA TGGATCCGTT 40 SEQ ID NO: 2 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence Characteristic of the symbol: Character-site location: 4..9 Method that determined the feature: S Symbol that represents the characteristic: cleavage-site Location: 18..23 Method that determined the characteristic: Symbol that represents the S feature : Cleavage-site Location: 32.37 Characteristic method: S sequence AACGAATTCA TCGAGGGTCG CGACCCTCG A TGAATTCGTT 40 SEQ ID NO: 3 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a procedure for preparing an expression vector in which the base sequence of Xa linker DNA is fused with the linker, which represents one example of the present invention.

FIG. 2 shows an example of the present invention, in which an expression vector containing Xa linker DNA is used as an exogenous gene for octopus rhodopsin 4
FIG. 2 is a view showing a procedure for preparing an expression vector in which a fragment gene is fused.

FIG. 3 is a diagram showing a procedure for preparing a 5 ′ end of a foreign gene fused to an expression vector containing Xa linker DNA.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshinori Harada 2520 Akanuma-cho, Hatoyama-cho, Hiki-gun, Saitama Prefecture Inside the Hitachi, Ltd. Basic Research Laboratory Co., Ltd. (56) References JP-A-61-135591 (JP, A) JP-A-2 -2386 (JP, A) JP-A-2-5881 (JP, A) "TaKaRa Reagents For Genetic Engineering Engineering Research, Japan, 1990, p. 28 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG)

Claims (12)

(57) [Claims] 1. An amino acid sequence recognized by blood coagulation factor Xa
Encoding Ile-Glu-Gly-Arg,
The codon in the Gly-Arg portion of the acid sequence is the sequence GGTC
GC, immediately after the 3 'end of the sequence GGTCGC.
Recognition of restriction enzyme Nru I with added sequence GA
The site TCGCGA has 14 base pairs formed,
Sequence C at the 3 'end by cleavage with the restriction enzyme Nru I
A smooth end having a G is formed.
Anchor DNA. 2. The factor Xa linker DNA according to claim 1,
The second character of the codon in the Arg portion of the amino acid sequence
At the blunt end having the sequence CG at
The blunt end at the 5 'end is A and T base pairs or G and C base pairs.
Xa linker DN characterized by being linked via
A. 3. The factor Xa linker DNA according to claim 1,
And restriction enzymes on both sides of the factor Xa linker DNA
Must have a recognition sequence or an end cleaved by a restriction enzyme.
Characteristic factor Xa linker DNA. 4. The factor Xa linker DNA according to claim 1,
And restriction enzymes on both sides of the factor Xa linker DNA
X having a recognition sequence or a cleavage end by a restriction enzyme
a Insert the linker DNA into the structural gene of the expression vector
And A and T base pairs or G and C base pairs are foreign genes.
The exogenous form formed just before the first codon of
The blunt end of the 5 'end of the gene or the base of A and T
The base or the base pair of G and C is the 5'-end of the foreign gene.
Added immediately before the start codon Met added to the smooth end
The blunt end of the formed 5 'end is the factor Xa linker
Ligation of DNA to the end cleaved by restriction enzyme Nru I
Expression vector characterized by having a fused gene
- 5. The factor Xa linker DNA according to claim 1,
And a restriction enzyme on both sides of the factor Xa linker DNA.
X having a recognition sequence or a cleavage end by a restriction enzyme
a Insert the linker DNA into the structural gene of the expression vector
And converting the factor Xa linker DNA into the restriction enzyme Nru.
Cleavage by I, A and T base pairs or G and C base pairs
Is added immediately before the first codon of the foreign gene.
The blunt end at the 5 'end of the foreign gene, or
A and T base pairs or G and C base pairs correspond to the foreign gene.
Immediately before the start codon Met added to the blunt end at the 5 'end
The blunt end at the 5 ′ end formed by adding
Characterized in that it is ligated to the end cleaved by the enzyme Nru I.
Method for producing a fusion gene. [6] A trait obtained by using the expression vector according to [ 4 ].
A live host cell characterized by being transformed. (7) A trait obtained by using the expression vector according to (4).
An Escherichia coli characterized by being transformed. 8. An amino acid sequence recognized by blood coagulation factor Xa
Encoding Ile-Glu-Gly-Arg,
The codon in the Gly-Arg portion of the acid sequence is the sequence GGTC
GC, immediately after the 3 'end of the sequence GGTCGC.
Recognition of restriction enzyme Nru I with added sequence GA
The site TCGCGA has 14 base pairs formed,
Amino acid sequence by cleavage with restriction enzyme Nru I
Has a sequence CG up to the second character of the codon of the Arg portion of
A base pair of A and T or a salt of G and C
A base pair is added immediately before the first codon of the foreign gene.
The blunt end at the 5 'end of the foreign gene
Means that the base pair of A and T or the base pair of G and C
Of the start codon Met added to the blunt end of the 5 'end of the offspring
The blunt end of the 5 'end formed by adding just before is connected.
Xa linker DNA characterized in that: 9. An amino acid sequence recognized by blood coagulation factor Xa
Encoding Ile-Glu-Gly-Arg,
The codon in the Gly-Arg portion of the acid sequence is the sequence GGTC
GC , immediately after the 3 'end of the sequence GGTCGC.
Recognition of restriction enzyme Nru I with added sequence GA
Xa with 14 base pairs where the site TCGCGA was formed
Linker DNA is inserted into the structural gene of the expression vector
And the restriction enzyme Nr of the factor Xa linker DNA
formed by cleavage with uI,
Has the sequence CG up to the second letter of the codon of the Arg portion
A and T base pairs or G and C base pairs at the 3′-end blunt end
Pair formed by adding just before the first codon of the foreign gene
A blunt end at the 5 'end of the foreign gene, or
A and T base pairs or G and C base pairs correspond to the foreign gene.
Immediately before the start codon Met added to the blunt end at the 5 'end
The blunt end of the 5 'end formed by adding to
It has a fusion gene and has a copy of the Arg portion of the amino acid sequence.
The base of the third character of don is any of A, T, G, and C
An expression vector, characterized in that: 10. An expression vector according to claim 9.
A living host cell characterized by being transformed. 11. An expression vector according to claim 9.
An Escherichia coli characterized by being transformed. 12. An amino acid sequence recognized by blood coagulation factor Xa.
Encoding the sequence Ile-Glu-Gly-Arg,
The codon in the Gly-Arg portion of the amino acid sequence is the sequence GGT
CGC, which is added immediately after the 3 ′ end of the GGTCGC.
Having the added sequence GA, and a recognition site for the restriction enzyme Nru I.
X having 14 base pairs where TCCGGA is formed
a Insert the linker DNA into the structural gene of the expression vector.
And insert the factor Xa linker DNA into the restriction enzyme Nr.
cleaved by uI to form the Arg portion of the amino acid sequence
Blunt ends with the sequence CG up to the second character of the codon
Formed at the 3 'end, A and T base pairs or G and C base pairs
Is added immediately before the first codon of the foreign gene.
The blunt end at the 5 'end of the foreign gene, or
A and T base pairs or G and C base pairs correspond to the foreign gene.
Immediately before the start codon Met added to the blunt end at the 5 'end
The blunt end at the 5 'end formed by adding
Ligating to the blunt end with CG to create a fusion gene
And the third letter of the codon in the Arg portion of the amino acid sequence
Is a base selected from the group consisting of A, T, G, and C.
Method for producing a fusion gene.